The high prevalence of nosocomial infections by multidrug-resistant Enterococcus faecalis presents both a treatment and an infection control challenge in the hospital setting. There is a clear demand to control enterococcal multi-drug resistance problems using newer approaches. Eukaryotic-type ser/thr-kinase (ESTK) and phosphatase (ESTP) are the recently recognized prokaryotic regulatory proteins that control the bacterial growth, division, metabolism, and expression of bacterial surface proteins, capsule, toxins, and virulence factors. Although information on the complete genome sequence of vancomycin-resistant strain of E. faecalis is recently available, there is no information on the nature and function of enterococcal ESTK (efSTK) and ESTP (efSTP). As a result, the importance of these proteins in enterococcal virulence and metabolism in general has not been appreciated. Based on the findings of group A Streptococcus (GAS) ESTK and ESTP that we recently characterized, we hypothesize that enterococcal efSTK and efSTP have functions akin to those described for GAS and play an important role in the regulation of growth, metabolism, expression of extracellular matrix-binding surface proteins, and toxins, which are important in the development of enterococcal biofilms and causation of endocarditis. To test this hypothesis, the contranscribing efstk and efstp genes will be cloned and corresponding recombinant proteins will be made to study their biochemical properties. To understand the biological functions of these putative signaling molecules, efSTK-specific knockout mutants will be created from three different E. faecalis strains with distinct pathogenic/genetic profiles. The impact of the deletion of this gene on bacterial growth, morphology, cell division, ability to adhere to HepG2 cells, and formation of biofilms on abiotic surfaces will then be determined using several laboratory assays. Using qRT-PCR assays, the role of efSTK in the regulation of the expression of selective genes that are responsible for the bacterial adherence, development of biofilms and virulence, will be determined. Finally, the impact on the enterococcal virulence will be elucidated by determining the ability of efSTK mutant to cause peritonitis in mouse and endocarditis in the rat animal infection models. The results obtained from this proposal will allow us to understand the role of these eukaryotic-type signaling proteins not only to better understand the mechanisms of enterococcal pathogenesis but also to help formulate strategies to develop novel chemotherapeutic agents against E. faecalis. Knowledge obtained from (i) the biochemical properties of enterococcal ESTK (efSTK) and ESTP (efSTP), (ii) phenotypic characteristics of isogenic knockout enterococcal mutants, (iii) qRT-PCR-based expression profiling of selected regulatory genes in these mutant and (iv) virulence profiling of these mutants in animal infection model will allow better understanding of the regulatory roles of these proteins in enterococcal pathogenesis, enterococcal multi-drug resistance, and to develop alternative chemotherapeutic strategies.